The association between Annexin A2 and epithelial cell adhesion molecule in breast cancer cells

Abstract Background The epithelial cell adhesion molecule (EpCAM) is a type I transmembrane and glycosylated protein, which is overexpressed in many neoplasms. However, EpCAM has no known ligand partners and the mechanisms by which it functions are not fully understood. Aim This study was performed to discover novel partners of EpCAM, which may provide a better understanding of its functions. Methods The membrane fraction of the ERα+ noninvasive breast cancer cell line ZR‐75‐1 and MCF‐7 was extracted and followed by co‐immunoprecipitation of EpCAM using C‐10, a mouse monoclonal antibody raised against amino acids 24–93 of the EpCAM molecule. As a negative control, MDA‐MB‐231 and Hs578T were used since they express a negligible amount of EpCAM and are known as EpCAM−/low ERα−/low invasive and tumorigenic breast cancer cell lines. Results Annexin A2 (ANXA2) was found to be selectively and differentially co‐immunoprecipitated with EpCAM in the ERα+ breast cancer cells MCF‐7 and ZR‐75‐1. ANXA2 is a multifunctional protein and known to act as a co‐receptor for tissue plasminogen activator (tPA) on the surface of endothelial and cancer cells, thereby affecting fibrinolytic activity and neoangiogenesis as well as invasive and metastatic properties. In this study, the association between EpCAM and ANXA2 was found to affect the activity of tPA. Conclusion This study concludes that ANXA2 co‐localizes with EpCAM at the plasma membrane, and the co‐localization may have functional implications. Data suggest that EpCAM supports ANXA2 to function as a co‐receptor for the tPA, and that EpCAM has a regulatory function on the expression and subcellular localization of ANXA2.

neoplasms including high grade glial tumors, 1 breast cancer, 2 and colorectal cancer. 3 However, there are complicated roles for EpCAM in the different cancers which may be attributed to the fact that the binding partners of EpCAM and the mechanisms by which it signals in and out of the cell are not fully known. 4,5 Therefore, EpCAM coimmunoprecipitation was performed and followed by mass spectrometry and peptide sequencing to search for new potential binding partners of EpCAM. The endoplasmic reticulum aminopeptidase 2 (ERAP2) has been identified by our group as an EpCAM-associated protein, 6 and here we continue this effort and present another novel finding that Annexin A2 (ANXA2) is a potential interacting partner of EpCAM in the EpCAM + ERα + breast cancer cells.
Annexins are a family of proteins with more than 22 members that have been isolated from a variety of cells and tissues and are involved in diverse physiological activities. Annexins consist of a variable amino terminal "tail" domain followed by four or eight conserved repeats. 7 Annexins in general bind to biological membranes and anionic phospholipids in a Ca 2+ dependent manner through these conserved repeats. ANXA2 belongs to this family of membrane binding proteins, and it controls apical plasma membrane and lumen formation. 8 The protein may cross-link plasma membrane phospholipids with actin and the cytoskeleton and be involved in exocytosis, membrane and vesicular trafficking.
ANXA2 was discovered by many investigators independently and given a variety of names (p34, p36, p39, calpactin I heavy chain, protein I, chromobindin-8, lipocortin II and placental anti-coagulant protein IV). 9,10 The protein exists in two major forms in cells, as a heterotetramer and as a 36 kDa monomer. A heterotetramer of ANXA2 contains two light chains of S100A10/p11 and two heavy chains of AnxA2/p36 and localizes to the cell surface. 11,12 The tetramer exists in the sub-plasma lemmal cytoskeletal network in different cell types. 13,14 As a monomer, ANXA2 is found in both the cytosol and nucleus, but predominantly in the cytosol. 15,16 The function of the ANXA2 monomer in the nucleus was suggested by its purification as part of a primer recognition protein complex that enhances DNA polymerase α activity in vitro. [17][18][19] Through S100A10, ANXA2 has been reported to function as a co-receptor for tissue plasminogen activator (tPA) at the cell surface. 20 In a previous report, it was shown that depletion of ANXA2 in telomerase immortalized microvascular endothelial cells led to the loss of plasminogen binding and plasmin generation similar to when S100A10 was depleted. 21 Furthermore, analysis of AnnexinA2-null mice showed that tPA-dependent plasmin generation at the endothelial cell surface is markedly deficient in the absence of ANXA2. 22 The phenotype of these mice showed that ANXA2 is a regulator of cell surface plasmin generation and that impaired endothelial cell fibrinolytic activity constitutes a barrier to effective neoangiogenesis.
The tPA is one of the proteases that convert the plasminogen to the active plasmin, and plasmin is involved in the degradation of the extracellular matrix. 23 The degradation of the extracellular matrix is a reported mechanism for progression of cancer, invasion and metastasis. 24 This study presents a novel finding that ANXA2 is an interacting partner of EpCAM in the EpCAM + ERα + breast cancer cells, and co-localizes with EpCAM at the plasma membrane of EpCAM + ERα + breast cancer cells. This co-localization may have a functional background since EpCAM appeared to support ANXA2 to function as a co-receptor for the tPA, and EpCAM seemed to have a regulatory function on the expression of ANXA2. were used in two previous publications from our group. 6,26 In those studies we could confirm their expected expression patterns of several breast cancer proteins as well as their phenotypes.
2.2 | Subcellular fractionation and EpCAM co-immunoprecipitation followed by gel staining, band picking and mass spectrometry 2.2.1 | Subcellular fractionation MCF-7, ZR-75-1, MDA-MB-231 and Hs578T cells were grown on 6-well plates up to 70%-80% confluence, and each cell line in duplicate. Then the cells were washed three times on ice with cold PBS and scraped off, cells were then spun down for 5 min at 700 G, 4 C.
Cell pellets were re-suspended in homogenization buffer (250 mM sucrose, 10 mM Tris-HCl buffer, pH 7.4). Homogenization was performed by drawing and releasing the suspended cells many times using 22 G needle and 10 cc syringe and the material was then spun down for 15 min at 1500 G, 4 C. The supernatant constitutes cytosolic proteins and membrane proteins, and it was then transferred to Sorval™ and centrifuged 10 600 rcf for 1 h at 4 C. The supernatant represents cytosolic fraction while the pellet is membrane fraction.

| Immunoprecipitation
The membrane fraction of the MCF-7, ZR-75-1, MDA-MB-231 and Hs578T cells was solubilized in ice-cold RIPA buffer 1:10 volume (RIPA buffer: 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0, 1% Non-Idet P-40/NP-40), and the suspension was centrifuged at 16 000 rcf for 15 min at 4 C. To clear any unbound immune complexes, the supernatant was incubated with protein G agarose beads at 4 C for 2 h (Immunoprecipitation starter pack, GE Healthcare), beads were spun down by centrifuging for 3 min at 3000 rcf and the supernatant was incubated with mouse monoclonal anti-human EpCAM (C-10, Santa Cruz) at a dilution of 2 μg per 100-500 μg of total protein (in 1 mL of cell lysate) overnight at 4 C. Immune complexes were captured with protein G agarose beads for 2 h at 4 C, eluted with 50 μL 2Â SDS sample buffer, shaken at 95 C for 10 min and centrifuged. The resulting supernatant represents EpCAM with possible co-immunoprecipitated proteins.

| qPCR using SYBR Green
The qPCR was done using the 2XSYBR Green Master Mix Kit (Applied Biosystem), and all the primers were obtained from Invitrogen (

| Western blot
After the transfection of siRNA and control, proteins were extracted using radioimmunoprecipitation assay (RIPA) buffer, and the amount of the protein quantified according to a standard assay protocol (DC protein assay, Bio-Rad, USA). About 50 μg of each sample prepared with SDS-loading buffer and proteins were separated on NUPAGETM 4%-12% Bis-Tris protein gels (Invitrogen). Each sample from each group (n = 4) were run in duplicates. Then, proteins were transferred to PVDF membranes using Trans-Blot Turbo transfer system (Bio-Rad). Membranes were blocked with 5% non-fat milk in PBS and incubated with the primary antibodies (

| Statistical analyses
All results were generated from at least two independent experiments. Data were analyzed using the Prism Graph Pad Software (CA, USA). All values are presented as mean ± SD. Significances were calculated using analysis of variance (ANOVA), followed by multiple comparisons. The values of p <.05 were considered significant whereas * indicates p-value <.05, **p-value <.01 and ***p-value <.001.
T A B L E 1 The primers used during the assessment of the relative expression levels

| Co-immunoprecipitation of ANXA2 with EpCAM in breast cancer cells
The gel used to separate the proteins after immunoprecipitation and from which slices were sent for the mass spectrometry is provided as a Figure S1 which was published as part of our coauthor's doctoral thesis. 27 Additionally, in the mass spectrometry analysis of ZR-75-1 cells, several proteins were found co-immunoprecipitated with EpCAM. Peptide finger printing was done, matched with NCBI protein database, Swissprot and ExPASy. Those proteins showing a statistically significant score are listed in Table 3. ANXA2 was identified with a score of 163/66 in the EpCAM + ZR-75-1 cell line (Tables 3 and 4), while it was not picked up by EpCAM co-immunoprecipitation followed by mass spectrometry analysis in MDA-MB-231 cells in which EpCAM is known to be almost absent (not shown). To confirm this finding, we first identified ANXA2 as a 38 kDa protein present at similar levels in ERα + and ERα À breast cancer cells (Figure 1(C)). Then EpCAM co-immunoprecipitation was repeated, and the precipitates were analyzed by Western blotting using ANXA2 antibodies. The reciprocal co-immunoprecipitation was performed using ANXA2 antibodies followed by Western blot analysis of EpCAM in the precipitates. This confirmed association between EpCAM and ANXA2 in ZR-75-1 cells (Figure 1(D),(E)).

| Co-localization of ANXA2 and EpCAM in breast cancer cells
Double immunofluorescence staining was performed to determine the subcellular localization of the two proteins. ANXA2 (green) displayed cytoplasmic as well as nucleolar staining patterns in all tested breast cancer cell lines, both in ERα + and ERα À cells (Figure 2(A)). EpCAM co-immunoprecipitation followed by Western blotting using ANXA2 antibodies confirmed an association between the two proteins in the ERα+ breast cancer cell line ZR-75-1. Notice that EpCAM appears as a band around 40 kDa while ANXA2 is slightly smaller, with a band at 38 kDa. This study was performed twice and independently, and plain beads without antibodies were added to the extract as a negative control However, ANXA2 in ERα + cells was clearly distributed at the plasma membrane more than in the cytoplasm and the nucleus in which it had a dot-like appearance; while in ERα À cells ANXA2 appeared diffusely in cytoplasm and nucleus (Figure 2(A)). EpCAM (red) showed membranous localization in MCF-7, ZR-75-1 and a subset of MC2 cells but was not detected in MDA-MB-231 and Hs578T cells.
ANXA2 and EpCAM co-localized as a line or dots, mainly at the plasma membrane of the ERα + cells (Figure 2(A)). Also, peripheral lamellae and membrane ruffles displayed ANXA2-EpCAM colocalization as illustrated in MCF-7 (Figure 2(B)), in addition to the were closely attached to each other. In MC2 cells, which are originally MDA-MB-231 cells engineered to express ERα, 28 ANXA2-EpCAM co-localization was also seen more diffusely in lamellae, at the periphery of cells (Figure 2(C)). In the MC2 cultures where only a subset of cells expressed EpCAM, ANXA2 tended to be localized only in the cytoplasm of EpCAM À cells, while also at the cell periphery of EpCAM + cells (Figure 2(D)).
F I G U R E 3 Legend on next page. The signals of ANXA2 and EpCAM were lost upon siRNA-ANXA2 and siRNA-EpCAM treatments, respectively. In comparison to the control, the signal intensity of EpCAM was weaker due to siRNA-ANXA2. However, due to siRNA-EpCAM treatment, the localization and signal of ANXA2 were almost lost to a level comparable to when ANXA2 was targeted by siRNA-ANXA2. (B) Effects of siRNA-ANXA2 or siRNA-EpCAM on ANXA2 and EpCAM by double immunofluorescence staining of ZR-75-1: The signals of ANXA2 and EpCAM were lost due to siRNA-ANXA2 and siRNA-EpCAM treatments, respectively. The signal intensity of EpCAM was weaker due to siRNA-ANXA2, compared to the control. Due to siRNA-EpCAM treatment, the localization and signal of ANXA2 were almost lost as if it was treated by siRNA-ANXA2 3.3 | The mRNA, protein levels and co-localization of ANXA2 and EpCAM in breast cancer cells after silencing EpCAM and/or ANXA2 Using siRNA against ANXA2 (siRNA-ANXA2) (Figure 3(A)) and EpCAM (siRNA-EpCAM) (Figure 3(B)) in MCF7 and ZR-75-1 resulted in a significant reduction of their respective mRNA levels, compared to the control groups ( Figure 3(A),(B)). In the ERα À/low and EpCAM À/low cells, the mRNA levels of ANXA2 were suppressed significantly upon siRNA treatment, in comparison to the control group treated with negative control siRNA ( Figure S2(A)). Noteworthy, the significant reduction in the mRNA levels of ANXA2 due to siRNA-ANXA2 was accompanied by a significant reduction in ANXA2 due to siRNA-EpCAM (Figure 3(A)). Moreover, the significant suppression of the mRNA levels of EpCAM due to siRNA-EpCAM treatment was paralleled by significantly suppressed levels of EpCAM due to siRNA-ANXA2 (Figure 3(B)).

| DISCUSSION
The binding partners of EpCAM and the mechanisms by which EpCAM signals in and out of cells have been extensively studied but are not fully known. Therefore, the current study used EpCAM which was previously reported to associate with EpCAM. 29 For confirmation of protein co-localization, we selected proteins that may contribute important novel functional mechanisms if a direct or indirect association with EpCAM can be confirmed. ERAP2 was identified by our group in a previous publication, 6 and here we chose to study ANXA2 further. This is because the association between ANXA2 and EpCAM, and the biological/functional significance of this association are presently unknown.
EpCAM protein, as our group reported previously, 6 is formed of 314 amino acids, and there are three domains: extracellular (the largest), transmembrane and cytoplasmic domain (Figure 1(A)). The ANXA2 protein is formed of 339 amino acids, and the amino acid sequence is shown in the Table 5. In the N-terminal domain, the domain for S100A10/p11 binding as well as known sites for regulatory tyrosine phosphorylation by pp60src and serine phosphorylation by PKC are all localized (Figure 1(B)). The assessment of the protein levels of ANXA2 and EpCAM relative to GAPDH showed that ANXA2 is expressed in all breast cancer cell lines whilst EpCAM is expressed only in the ERα + cells ZR-75-1 and MCF-7 (Figure 1(C)). The latter observation is consistent with what has been reported previously. 4,30 Double immunofluorescence stains on EpCAM + cells showed that the two proteins co-localized mainly in a membranous pattern.
Using siRNA-ANXA2, the signal intensity of EpCAM was weaker.
However, by using siRNA-EpCAM treatment, the localization and signal of ANXA2 was almost lost to a level comparable to when ANXA2 was targeted by siRNA-ANXA2 (Figure 4(A),(B)). Regarding the latter observation, it was thought that losing the localization and signal of ANXA2 due to siRNA-EpCAM might be related to the secretion of ANXA2 to extracellular space or medium. 31 10  20  24  26  30  40  50  60   MSTVHEILCK  LSLEGDHSTP  PSAYGSVKAY  TNFDAERDAL  NIETAIKTKG  VDEVTIVNIL   70  80  90  100  110  120   TNRSNAQRQD  IAFAYQRRTK  KELASALKSA  LSGHLETVIL  GLLKTPAQYD  ASELKASMKG   130  140  150  160  170  180 LGTDEDSLIE of the unbound tPA, the ERα À/low and EpCAM À/low cells, MDA-MB-231 and Hs578T, were included as a control. The results showed that the concentration of tPA in the cell culture supernatants of MDA-MB-231 and Hs578T increased significantly due to siRNA-ANXA2 treatment ( Figure 5(C)). This is consistent with a previous study, which has assessed tPA after silencing of ANXA2 in MDA-MB-231. 33 It is known that ANXA2 has diverse functions, and it is involved in cancer cell motility, invasion, and metastases. [34][35][36][37][38] Moreover, the up-regulation of ANXA2 expression in cancer was directly correlated with advanced clinical stage 39,40 ; and higher ANXA2 expression was also observed in metastatic breast and colon cancer cells compared with the non-metastatic cells. 36,41 All of these different roles of ANXA2 were linked in different reports to ANXA2-tPA-dependent plasmin generation. 20,32,33,42 In conclusion, the current study presents novel findings that may add insights for a better understanding of the complicated roles of EpCAM and ANXA2, which have been investigated and studied separately for decades. These findings include (1)